Field of the Invention
The present disclosure in general relates to an expression system. More particularly, the present disclosure relates to an expression system for the production of a protein that has a N-terminal pyroglutamate (pGlu) residue.
Description of Related Art
Cyclization of a glutaminyl or glutamyl residue to form a pyroglutamate (5-oxoproline, pGlu) residue occurs at the N-terminus of numerous secretory proteins and peptides. The N-terminal pGlu modification is known to protect the proteins and peptides from exopeptidase degradation, and endow them with a proper conformation for binding to their receptors. To date, a large number of proteins and peptides with N-terminal pGlu modification have been reported, including thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), neurotensin, gastrin, glucagon, monocyte chemoattractant protein (MCP), immunoglobulin, and ribonuclease.
The N-terminal pGlu formation on proteins and peptides are catalyzed by glutaminyl cyclase (QC). Two types of QCs have been reported. Type I QCs display a five-bladed β-propeller fold and are mainly identified in plants, pathogenic bacteria, and human parasites; and type II QCs adopt an α/β topology and are abundant in the neuroendocrine tissues and peripheral blood lymphocytes of mammals. Within the mammalian cells, QCs are mainly identified in the secretory granules and Golgi apparatus, where majorities of secreted hormones and chemokines are present.
Tobacco etch virus protease (TEVP), a cysteine protease, recognizes and exhibits high cleavage efficacy toward the sequence EXLYφQ\φ, in which X is any residue, φ is any large or medium hydrophobic residue, and φ is any small hydrophobic residue. Based on the cleavage specificity, TEVP is usually used as a biological tool to remove the tag protein that is linked with a target protein by the TEVP recognition sequence. However, TEVP is insoluble in water; thus, a carrier protein that exhibits the ability to prevent protein aggregation, such as the maltose-binding protein (MBP) and the N-utilizing substance A (NusA), is generally needed to be co-expressed with TEVP so as to increase its solubility.
Generation of N-terminal pGlu-modified proteins by either Escherichia coli (E. coli) or eukaryotic cells is a challenging work, due to the fact that the fusion protein cannot be recovered without the following two-step reaction: (1) the removal of tag protein by protease to expose the N-terminal glutaminyl or glutamyl residue, and (2) converting the exposed glutaminyl or glutamyl residue into a pGlu residue, a cyclization reaction catalyzed by QC. The two-step reaction not only limits the production efficiency, but also substantially reduces the product yield.
In view of the forgoing, there exists in the related art a need for an improved expression system and/or method that produce N-terminal pGlu-modified proteins without performing the conventional two-step reaction.